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Published online 10 November 2011
Nucleic Acids Research, 2012, Vol. 40, No. 5 1891–1903
doi:10.1093/nar/gkr681
SURVEY AND SUMMARY
Self-assembled nucleolipids: from supramolecular
structure to soft nucleic acid and drug
delivery devices
Vanessa Allain, Claudie Bourgaux* and Patrick Couvreur
Laboratoire de Physicochimie, Pharmacotechnie et Biopharmacie, UMR CNRS 8612, Université Paris-Sud 11,
Faculté de Pharmacie, 5 rue J.B. Clément, 92296 Châtenay-Malabry, France
Received June 10, 2011; Revised July 29, 2011; Accepted August 2, 2011
ABSTRACT
This short review aims at presenting some recent
illustrative examples of spontaneous nucleolipids
self-assembly. High-resolution structural investigations reveal the diversity and complexity of assemblies formed by these bioinspired amphiphiles,
resulting from the interplay between aggregation
of the lipid chains and base–base interactions.
Nucleolipids supramolecular assemblies are promising soft drug delivery systems, particularly for nucleic
acids. Regarding prodrugs, squalenoylation is an innovative concept for improving efficacy and delivery
of nucleosidic drugs.
INTRODUCTION
Hybrid molecules composed of a lipid covalently linked to
a nucleoside have been identified in both eukaryotic and
prokaryotic cells. The most common of these nucleolipids,
cytidine diphosphate diacylglycerol, is a key intermediate
of the biosynthesis of glycolipids and lipoproteins. It may
also be involved in the synthesis of phosphatidylinositol
and cardiolipin, two components of mammalian cell membranes. Other naturally occurring nucleolipids, isolated from
bacteria (tunicamycins, liposidomycins and septacidin) or
marine sponges (agelasines), which elicit biological responses such as antimicrobial, antifungal, antiviral or antitumor activities, have also been reviewed by Rosemeyer (1).
Many molecules currently used in clinic against cancer
or viral infections such as AIDS are nucleoside analogues.
For example, Gemcitabine and Zalcitabine are derived
from cytidine, while Didanosine is derived from adenosine
and Zidovudine (AZT) from thymidine. Their main mechanism of action is a potent inhibition of DNA or RNA
synthesis, which may lead to cell cycle blockage and eventually apoptosis. However, their therapeutic potential is
often restricted by a poor stability in vivo, the induction
of severe side effects and a limited passive intracellular
diffusion due to their hydrophilicity. Nucleoside analogues enter cells mainly through specific membrane transporters, whose inactivation can result in resistance.
Various lipophilic derivatives of nucleoside analogues have
thus been designed in an attempt to circumvent these
drawbacks. Site-specific bioconjugation enables to protect
sensitive groups from enzymatic degradation and the
coupling of hydrophobic moieties may yield amphiphilic
conjugates able to cross the plasma membrane by passive
diffusion.
Beyond prodrugs, the interest in nucleolipids was further
stimulated by the self-organization properties of these
amphiphilic molecules combining the aggregation characteristics of lipids and the specific functionalities of nucleosides. Self-assembly in an aqueous medium of amphiphiles
possessing both a polar head derived from a nucleobase
and a lipidic moiety offers the possibility to obtain new
supramolecular systems with original biophysical properties for numerous applications ranging from biomaterials
to drug delivery systems. Supramolecular devices formed
by nucleolipids are promising carriers for nucleic acids
delivery. Indeed, nucleotides being the constituting monomers of nucleic acids, it is expected that base–base interactions between nucleic acids and nucleolipids can ensure
the entrapment of DNA, RNA or oligonucleotides in
these systems. In an effort to expand the field of this
family of lipids, numerous molecules have thus been recently synthesized and their supramolecular organization–
properties relationship investigated.
The purpose of this short review is to present recent
examples in the field of spontaneous nucleolipids selfassembly, focussing on the structures formed and their
possible therapeutic applications. The interactions between
nucleolipids are first presented, with emphasis on molecular recognition properties between complementary bases.
*To whom correspondence should be addressed. Tel: 33 1 46 83 56 44; Fax: 33 1 46 83 53 12; Email: [email protected]
ß The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
1892 Nucleic Acids Research, 2012, Vol. 40, No. 5
The methods most often used to study the structure of
self-assembled nucleolipids are recalled. In the following
part, examples of hierarchical architectures formed by
nucleolipids reveal how aggregation of the lipid moieties
interferes with interactions involving nucleobases. The use
of nucleolipid-based devices for nucleic acids transfection
is then presented. Finally, we describe lipidic derivatives of
nucleosidic drugs that self-organize as nanoparticles. A
new strategy, known as ‘squalenoylation’, has been
recently conceived in our laboratory. It involves the coupling of squalene, a natural precursor in the biosynthesis of
sterols, to nucleoside analogues such as Gemcitabine or
Zalcitabine. These derivatives exhibited impressive higher
anti-cancer or antiviral activity than the parent drugs.
Remarkably, the amphiphilic molecules obtained selforganize into nanoassemblies in aqueous medium, whatever the nucleoside analogue. Squalenoylation provides an
original platform for improving efficacy and delivery of
nucleosidic drugs, which could be extended to other polar
therapeutic compounds.
INTERACTIONS BETWEEN NUCLEOLIPIDS:
MOLECULAR RECOGNITION AND
SELF-ASSEMBLY
The self-assembly of complex structures, from similar or
different components interacting by multiple non-covalent
intermolecular forces, is ubiquitous in biology, soft condensed matter, and nanoscience. The resulting supramolecular ordered assemblies usually exhibit a hierarchy
of structural levels. Weak dynamic bonds may impart
stimuli-responsive and time-dependent properties to the
aggregates. For instance, nanoparticles could alter their
structure in response to an external stimulus such as pH
or temperature change (2).
The self-assemblies of lipid-nucleoside (or nucleotide)
conjugates are typical examples of such ordered stimuli
responsive structures. These nucleolipids possess a polar
head derived from either a purine base (adenine or guanine) or a pyrimidine base (cytosine, thymine or uracil).
These bases can interact through -stacking and
hydrogen bonding, each base–base motif displaying different binding characteristics (3). The base pairs present in
nucleic acids are adenine–thymine and guanine–cytosine
in DNA double strands or adenine–uracil and guanine–
cytosine in RNA double strands. Each strand of DNA (or
RNA) is coupled through H-bonding to the other strand
formed by the complementary bases sequence. These interactions being weak, base pairing is a cooperative process
assisted by the macromolecular nature of nucleic acids. It
has been evidenced that simple nucleolipids in micellar or
vesicular aggregates, or adsorbed on a surface, could display
similar molecular recognition properties between complementary bases (4–10). For example, in mixed micelles
formed at physiological pH from the equimolar mixture of
two phospholipids functionalized with either uridine or adenosine, i.e. dioctanoylphosphatidyl-adenosine (diC8PA)
and dioctanoylphosphatidyl-uridine (diC8PU), a specific
molecular recognition process was identified at the micellar
surface. Both stacking and H-bonding between adenosine
and uridine bases were evidenced by UV and circular dichroism (CD) spectra and 1H NMR measurements. These
interactions induced a decrease of the mean area per polar
headgroup, compared to that of the pure components in
individual micelles (5,6).
It should be emphasized that H-bonding is not a sufficient driving force for molecular recognition between complementary bases in water. Below the critical micellar
concentration (cmc), molecular recognition is absent in
monomeric solutions of nucleolipids bearing complementary headgroups. Likewise, stacked structures of isolated
nucleosides are more stable than hydrogen-bonded ones in
water. Anchoring of at least one of the bases to an
organized interface seems to be a prerequisite for molecular recognition, highlighting the importance of the cooperative effect.
Identical nucleolipids can also interact. The interplay
between supramolecular organization of nucleolipids and
base–base interactions has been underlined. Stacking and
H-bonding of the headgroups are expected to depend on
the structure of the aggregates through the confinement
induced by aggregation. The interfacial curvature of aggregates may further influence the efficiency of the interactions. In turn, base–base interactions can themselves
modulate the supramolecular structure of nucleolipids.
Small differences between polar headgroups may cause
variation in the phase behaviour. Regarding the lipid moiety, beside the number, length and degree of unsaturation
of hydrophobic chains, their position relative to the base
can have an impact on the formed structure. As a result,
self-assembled nucleolipids may exhibit a variety of structures. Vesicules, lamellar phases, ribbons, helical strands,
spherical or wormlike micelles, hexosomes or cubosomes,
etc., have been identified (9,11–15).
METHODOLOGIES USED FOR INVESTIGATING
NUCLEOLIPIDS SUPRAMOLECULAR ASSEMBLIES
Self-assembled nucleolipids are organized at different
length scales. Their morphology and structure have been
investigated by transmission electron microscopy (TEM),
X-rays, neutrons and light scattering while spectroscopic methods, mainly UV and CD spectroscopy and
1
H NMR measurements, gave an insight into base–base
interactions. Whereas TEM allowed a direct imaging of
nucleolipids-based nanoassemblies, the characteristic
lengths of the supramolecular structures (ranging from
atomic distances up to hundreds of nanometres) may be
probed by X-ray (small- and wide-angle X-ray scattering,
SAXS–WAXS) and small-angle neutron scattering
(SANS). WAXS describes the structure at an atomic
scale (from 1 to 10 Å), whereas SAS is related to the
supramolecular organization. SAXS allows measuring
characteristic dimensions between 10 Å and 1000 Å,
whereas larger length scales can be reached with SANS.
Generally, the symmetry of ordered phases (lamellar,
cubic, hexagonal, etc.) results in Bragg reflections whose
positions are in characteristic ratios. In the case of a
micellar phase, without long-range order, the SAS
pattern contains information on the size and shape of
Nucleic Acids Research, 2012, Vol. 40, No. 5 1893
the micelles and on their mutual interactions within the
solution. Noteworthy, the availability of Synchrotron radiation (SR) sources with high brightness and high collimation enlarges the possible uses of X-ray scattering for
the investigation of large structural features and dilute, or
weakly scattering, samples. In view of the lability of the
interactions between the molecular components of a
supramolecular entity, the structure of nanoparticles could
change as a function of temperature. Microcalorimetry,
able to determine with high sensitivity the thermal exchanges involved in modifications of the nanoassemblies,
can indicate phase transitions. When DSC is coupled with
SAXS, a detailed phase diagram can be established. The
UV and CD spectra of bases, in the 180–300 nm range, are
sensitive to base–base interactions. Stacking of bases gives
rise to a hypochromic effect accompanied by an increase
of the molar ellipticities. Hydrogen bonding between bases
can be assessed by FTIR spectroscopy and 1H NMR.
H-bonding causes a downfield shift of the resonances of
the base protons, while stacking induces an upfield shift.
The use of these techniques to elucidate the supramolecular structures of self-assembled nucleolipids is illustrated
by examples below.
EXAMPLES OF HIERARCHICAL ARCHITECTURES
FORMED BY NUCLEOLIPIDS
Advantage may be taken from the interactions between
nucleolipids to construct original materials. In this view,
non-ionic, zwitterionic, anionic and cationic nucleolipids
have been synthesized. Examples of chemical structures of
nucleolipids belonging to the main groups of synthetic bioconjugates are given in Figure 1. Their self-organization
gave rise to a diversity of aggregate morphologies.
Baglioni and co-workers have synthesized nucleolipids
by exchanging the choline headgroup of phosphatidylcholines with a nucleoside being either uridine or adenosine.
The lipidic moiety was attached on 50 -position of the nucleoside. The obtained molecules had a nucleotide group,
bearing a negative charge, as polar heads. Globular
micelles, flexible cylindrical aggregates and bilayers were
observed upon self-assembly of these nucleolipids, depending on the length of the alkyl chains used. Shortchain derivatives (C6–C9) formed quasi-spherical micelles
whereas long-chain derivatives adopted locally flat topologies, consistent with the increase in the packing parameter
with longer chains. 1-palmitoyl-2-oleoyl-phosphatidyluridine (POPU) and 1-palmitoyl-2-oleoyl-phosphatidyladenosine (POPA) (Figure 1G) formed bilayers (16,17).
Remarkably, 1,2-dilauroyl-phosphatidyl-uridine (DLPU)
was found to behave as an ‘associative polynucleotide’.
At physiological pH DLPU spontaneously self-assembled
into cylindrical aggregates whose length could be tuned by
the nucleolipid concentration and by the ionic strength of
the aqueous medium (Figure 2A). Increase in concentration in the presence of added salt or increase in ionic
strength at sufficient concentration entailed micellar unidimensional growth, as shown by a combination of scattering techniques and cryo-TEM. The cylindrical local
structure, with cross-sectional radius of 20 Å, remained
unchanged during micellar growth. Giant wormlike micelles, characterized by an apparent persistence length of
230 Å (in the 0.1 M PBS buffer), entangled to form a transient network with properties comparable to those of
semi-dilute polymer solutions. However, unlike polymers,
flexible micelles could break and recombine (18).
In general, the dominant factor determining these
supramolecular structures was the hydrophobic part of
the nucleolipids but, as emphasized, the nature of the
nucleobase modulated their aggregation behaviour. The
impact of the nucleobase was anticipated to be more
evident for DLP-nucleoside and POP-nucleoside derivatives than for diC8P-nucleoside derivatives because the
lower interfacial curvature of cylindrical or planar
nanoassemblies was expected to favour base interactions,
especially base stacking, in comparison to the high interfacial curvature exhibited by spherical micelles. Meaningful
differences between adenosine and uridine long-chain derivatives have, indeed, been stressed. The 1,2-dilauroylphosphatidyl-adenosine (DLPA) bioconjugate showed a
more complex self-assembly pattern than DLPU. DLPA
displayed a time-dependent evolution leading to the hierarchical aggregation of wormlike micelles initially formed.
The helical superstructures obtained were promoted by
stacking and H-bonding of adenosine moieties (19).
Likewise, the lamellar phase of POPA was characterized
by an higher main transition temperature Tm, a lower hydration and a less ordered stacking of bilayers, compared
to the lamellar phase of POPU. The above differences
have been explained by the different stacking and
H-bonding characteristics of adenosine and uridine. In
particular, purines, like adenine, have higher staking constants than pyrimidines, like uracil. Molecular modelling
further suggested that POPA molecules arranged preferentially in pairs. Tighter arrangement of adenosine groups
at the interface could result in the lower cross-sectional
area found for adenosine, although this derivative is
bulkier, and in partial water exclusion from the interfacial
region of bilayers (17).
Noteworthy, spontaneous formation of helical structures was evidenced by Yanagawa et al. (12) upon aging
at room temperature of dimyristoyl-50 -phosphatidyldeoxycytidine conjugate solution (Figure 2B). This conjugate first self-assembled as double helical strands with a
diameter of 110 Å and a helical pitch of 240 Å.
Superhelical structures with a helical pitch of 1000 Å
further appeared. Yanagawa et al. have then attempted
to clarify the conditions of helical structure formation by
varying the alkyl chains length and the nature of the nucleoside. They concluded that helical strands were stabilized by the balance between hydrophobic chains
interactions, depending on their length, and bases
stacking (13).
Barthelemy and co-workers have chosen to attach two
lipophilic chains to the 20 - and 30 -positions of the nucleoside or one chain to its 30 -position. In the first study, they
have synthesized a series of uridine-based zwitterionic
nucleolipids possessing two acyl chains on the 20 - and
30 -positions of the nucleoside and a phosphocholine group
on the 50 -position (20). The 1,2-dipalmitoyluridinophosphocholine (DPUPC) compound (Figure 1B), displaying two
1894 Nucleic Acids Research, 2012, Vol. 40, No. 5
Figure 1. Examples of chemical structures of main synthesized nucleolipids (except prodrugs). (A) Deoxythymidine 30 -palmitoylphosphate (C16-30 TMP)
(21); (B) 1,2-dipalmitoyluridinophosphocholine (DPUPC) (20); (C) 20 ,30 -dioleoyl)uridine-trimethylammonium tosylate (DOTAU) (28); (D) O-ethyl-dioleylphosphatidylcholinium-uridine (O-ethyl-DOUPC) (27); (E) 50 -PEG5000-20 ,30 -dilauroyl-sn-glycero-3-uridine (DLU-PEG5000) (31). (F) 1(20 ,30 -dioleylcarbamoyl-uridine-50 )--D-glucopyranoside (DOUGluc) (32). (G) 1-palmitoyl-2-oleoyl-sn-glycero-3-adenosine (POPA) (16); (H)
deoxythymidine-30 -(1,2-dipalmitoyl-sn-glycero-3-phosphate) (diC16-30 dT) (38).
saturated chains of 16 C, illustrates the interplay between
the organization of the lipid chains and base–base interactions. In aqueous medium, DPUPC self-assembled as
bilayers when the lipid chains were in the fluid phase
above the main transition temperature Tm and formed
fibres when they were in the gel state, below Tm. As
shown by SAXS and TEM, the fibres resulted from
hexagonal packing of helical strands (Figure 3). The UV
and CD spectra of DPUPC aggregates as a function of
temperature evidenced a different organization of bases in
the fibres and bilayers. The transition between the two
supramolecular structures was reversible. Above a threshold concentration (6% w/w), a hydrogel was stabilized by
a network of fibres, at temperatures below Tm.
Nucleic Acids Research, 2012, Vol. 40, No. 5 1895
Figure 2. Spontaneous formation of wormlike and helical micelles from phospholipid-nucleoside conjugates. (A) Cryo-TEM image of 10 mM DLPU
micellar solution in 0.1 M PBS at pH 7.5 showing wormlike micelles polydisperse in length [reprinted with permission from (19). Copyright (2006)
American Chemical Society]. (B) Electron micrograph of superhelical strands formed from dimyristoyl-50 -phosphatidyldeoxycytidine (DMPcyt) after
ageing at 25 C for 1 day [redrawn from Ref. (12)].
Figure 3. Illustration of the thermoreversible helical nanofibres, lamellar phase transition undergone by DPUPC in water. Proposed model for the
helical nanofibres (A and B): (A) two molecules of DPUPC forming the basic repeat unit of the helical structure shown in (B). (B) Drawing of one
helical strand (helical pitch 7.0 0.5 nm, diameter 4.2 0.5 nm). (C) Drawing of the multilamellar organization [reprinted with permission from (20).
Copyright (2004) American Chemical Society].
Later investigations have assessed the impact of the
nature of the nucleoside on the supramolecular structure
of nucleolipids bearing a single monophosphate palmitoyl
or eicosyl chain on the nucleoside 30 -position (Figure 1A)
(21). These nucleotide-based amphiphiles formed colloidal dispersions upon sonication in an aqueous medium.
They self-organized into ribbon-like nanoassemblies
for the thymidine derivatives and in aggregates of small
crystals for the adenine derivatives, as revealed by TEM.
Wide angle X-ray diffraction patterns evidenced crystallike 3D structural ordering of the dried aggregates.
FTIR spectra were indicative of extended alkyl chains
arranged in an orthorhombic lattice for the thymidine derivatives and displaying a triclinic packing for the adenine
derivatives. The structural ordering also arose from
hydrogen bonding between opposite bases and adjacent
bases stacking in the 3D lattices, as shown by FTIR
spectra.
1896 Nucleic Acids Research, 2012, Vol. 40, No. 5
Self-assembly of nucleotide-terminated bolaamphiphiles
has also been used to obtain hydrogels through spontaneous formation of a network of intertwined fibres. The
chain-end functionalization of long oligomethylene spacers
(C18 or C20) with 30 -phosphorylated thymidine moieties
yielded a hydrogel at concentration as low as 0.2 wt%.
Both hydrophobic interactions between the spacer chains
and interactions involving the nucleotide moieties were
responsible for the formation of nanofibres (22).
TOWARDS GENE THERAPY: NANOSTRUCTURED
NUCLEOLIPID ASSEMBLIES FOR NUCLEIC ACIDS
TRANSFECTION
The aim of gene therapy is to cure inherited or acquired
genetic disorder or diseases, including cancer, by replacing
or inhibiting the faulting gene in cells, using natural or
synthetic nucleic acids. The delivery to cells of DNA, antisense oligonucleotides or si-RNA, requires a vector such
as lipoplexes. The majority of lipoplexes consists of nucleic
acids mixed with cationic lipids in the presence of a
‘helper’ lipid, usually dioleoylphosphatidylethanolamine
(DOPE) or dioleoylphosphatidylcholine (DOPC). In this
approach, the negatively charged DNA interacts through
electrostatic forces with the positively charged lipid.
Structural studies have shown that DNA can be complexed
into lamellar or hexagonal lipid phases (Figure 4). The
addition of the ‘helper’ lipid favours the inverse hexagonal
phase, which has been suggested to enhance transfection.
The transfection efficiency of lamellar cationic lipoplexes
depends on the charge density of lipid membranes,
probably because of their electrostatic interactions with
cells. In the regime of optimal membrane charge density,
Figure 4. Schematic representation of the structure of lamellar and
hexagonal lipid–DNA complexes. The lamellar phase Lac exhibits
DNA rods intercalated between lipid bilayers. The hexagonal phase
HIc consists of DNA rods between rodlike lipid micelles arranged on
a hexagonal lattice. The inverse hexagonal HIIc phase consists of DNA
rods coated with a lipid monolayer arranged on a hexagonal lattice.
The lipids are in red (headgroup) and grey (chain) and the DNA rods
are in blue [redrawn from Ref. (61)].
maximum in transfection efficiency was obtained when
bilayers were made of a multicomponent lipid mixture,
highlighting the importance of carrier lipidic composition
(23). Unfortunately, after administration, the positive
charges of the cationic lipids were demonstrated to dramatically interact with serum proteins, resulting in their
fast elimination from the blood flow. Another drawback is
the common cytotoxicity of cationic formulations due to
cell membrane destabilization. Alternative delivery
systems, bearing fewer charges, are thus considered in an
attempt to overcome these problems. For instance, new
lipids containing in their polar head thiourea functions,
known as strong hydrogen bond donors, have been developed. Lipopolythioureas were capable of compacting
DNA thanks to multiple hydrogen bonds involving the
DNA phosphate groups. Moreover, the presence of the
three forms of the thiourea function (i.e. thiourea,
iminothiol and charged thiourea), afforded in vitro transfection of the lipids/ DNA complexes (24–26).
A new strategy, initiated by the research groups of
Barthelemy and Baglioni, relies on self-assembled nucleolipids to complex and transfect nucleic acids, taking advantage of base–base interactions between nucleolipids
and nucleic acids. In this approach, a series of cationic
nucleolipids derived either from uridine, like O-ethyldioleyl-phosphatidylcholinium-uridine (O-ethyl-DOUPC)
(Figure 1D) and 20 ,30 -dioleoyluridine-trimethylammonium
tosylate (DOTAU) (Figure 1C), or from a 3-nitropyrrole
universal base analogue were synthesized by Barthelemy
and co-workers. O-Ethyl-DOUPC and DOTAU interacted with both DNA double helix and polyadenylic
acid (polyA) single strand. TEM and SAXS revealed
multilamellar systems with a lattice of DNA rods
intercalated between lipid bilayers (27,28). O-EthylDOUPC at high nucleolipid/DNA ratios (18/1 and 36/1
w/w) displayed a good transfection efficacy in CHO cells,
comparable to that of the commercially available
Transfast, without cytotoxicity or inhibition of cell proliferation. Vesicles formed by cationic nucleolipid based on a
3-nitropyrrole universal base analogue were shown to efficiently transfect siRNA in three different cell lines (29).
The weakly hydrogen bonding 3-nitropyrrole is known to
form base pairs indiscriminately with the natural bases
when opposite them in an oligonucleotide duplex, thanks
to stacking interactions with adjacent bases (30).
Beside cationic nucleolipids, neutral amphiphiles derived
from uridine and possessing one or two alkyl chains and
either a glucose (Figure 1F) or a PEG (Figure 1E) moiety
have been shown to complex nucleic acids (31,32). The
rationale behind these chemical modifications of the headgroups was to impact the aggregation of the nucleolipids
and to reinforce the interactions between their supramolecular assembly and nucleic acids, by introducing additional interactions, such as phosphate–sugar interactions.
In particular, the compound displaying an oleyl chain, a
nucleoside and a glucose moiety linked together by
1,2,3-triazole bridges (GNL) was found to be a highly effective hydrogelator at concentrations >2.5% w/w. The
resulting gel was thermoreversible, melting upon heating
and turning back to gel on cooling. It was formed by an
entangled network of fibres of 20–30 nm in diameter, as
Nucleic Acids Research, 2012, Vol. 40, No. 5 1897
evidenced by phase contrast microscopy and TEM. SAXS
experiments, performed to unravel the structure of these
fibres, strongly suggested that interdigitated GNL molecules assembled as bilayers forming hollow nanotubes.
Base-stacking interactions within the bilayers were supported by UV measurements. The gel network was able
to entrap small condensed particles of oligonucleotides,
aggregated on the fibre surface. This nanostructured
material favoured the internalization of oligonucleotides
into cells, without significant toxicity (33). Recently,
fluorinated analogues of these glycosyl-nucleolipids have
been synthesized and their gelation properties studied (34).
The most challenging approach was to induce nucleic
acids complexation using anionic nucleolipids. In the formulation of anionic lipoplexes, consisting in the mixture
of anionic and zwitterionic phosphoplipids with DNA, the
presence of divalent cations, usually Ca2+, is critical to
achieve the complexation between DNA and the phospholipids (35). The addition of Ca2+ allows overcoming the
electrostatic repulsion between negatively charged lipids
and DNA. Divalent cations have also been shown to
mediate interactions between zwitterionic phospholipids
and DNA, bridging the phospholipid headgroups and
the DNA rods (36,37). Barthelemy et al. have described
a formulation involving a new anionic nucleolipid possessing thymidine-30 -monophosphate as polar head and
1,2-diacyl-sn-glycerol as lipid moiety, diC16-30 -dT (Figure
1H). Mixtures of DOPE and diC16dT complexed DNA in
the presence of Ca+2, which resulted in the formation of
both hexagonal and lamellar phases. This formulation was
non-toxic and revealed a good in vitro transfection
efficacy, likely owing to the presence of the inverted hexagonal phase (38).
Further, Baglioni and co-workers have provided the
first proof of concept that complexes of nucleic acids
and anionic nucleolipids can be stabilized via base pairing
only, without the presence of cations (39,40). A polyuridilic acid chain (polyU) was chosen as model of a
single strand of nucleotide homopolymer. This polynucleotide was shown to interact with either POPA or
diC8PA, two negatively charged nucleolipids bearing the
complementary adenine base. SAXS experiments have
evidenced well-ordered POPA/polyU complexes. PolyU
chains were intercalated between POPA fluid bilayers,
inducing a lamellar spacing increase. They formed a 1D
lattice, with a characteristic spacing depending on the
POPA/polyU molar ratio. PolyU also bound to diC8PA
spherical micelles, mediating the formation of clusters involving several polyU chains and micelles, as shown by
dynamic light scattering, SANS and SAXS. Interestingly,
these clusters showed a time-dependent evolution leading
to the formation of a well-defined hexagonal phase with a
lattice parameter of 98 Å. This supramolecular assembly
could be modelled as a hexagonal array of diC8PA cylindrical micelles templated by single strands of the complementary polynucleotide (Figure 5). For both POPA/
polyU and diC8PA/polyU systems, the selective interactions between the complementary bases of the polynucleotide and the nucleolipids, confirmed by spectroscopic
measurements, were the driving force for their supramolecular association. Thus, molecular recognition between
complementary bases is able to overcome electrostatic repulsion between like-charged polynucleotide and nucleolipids and to trigger structural re-organization of the
mixed system. Table 1 gathers nucleolipid/nucleic acid
complex devices displaying transfection efficacy in vitro
on various cell lines.
It should be mentioned that, beside lipoplexes, lipidconjugated oligonucleotides were also considered as an alternative strategy for the delivery of nucleic acids (41–43).
TOWARDS THE CONCEPTION OF
NANOMEDICINES: THE SELF-ASSEMBLED
NUCLEOSIDIC PRODRUGS
The prodrug strategy has been successfully explored to
improve the anti-cancer or antiviral efficacy of nucleosidic
analogues. In this approach, a hydrophobic moiety is usually linked to the nucleoside analogue to obtain an
Figure 5. Proposed model for the supramolecular structures formed by polyuridilic acid (polyU) mixed with dioctanoyl-phosphatidyl-uridine
(diC8PA) in 0.05 M Tris buffer (pH 7.5). Clusters of polyU chains and diC8PA micelles formed first. After storage at 4 C for one week a well
defined hexagonal phase was obtained, suggesting a hexagonal arrangement of diC8PA cylindrical micelles templated by strands of the complementary polynucleotide [reproduced with permission from Ref. (39)].
1898 Nucleic Acids Research, 2012, Vol. 40, No. 5
Table 1. Nucleolipid/ nucleic acid complexes displaying transfection (or internalisation) efficacy in vitro
Nucleolipids
Nucleic acids
Expression vectors
Nucleolipid/nucleic acid ratios
Cell lines
References
O-Ethyl-DOUPC
Plasmid DNA
18/1, 36/1 (w/w)
CHO
(27)
DOTAU
Plasmid DNA
2.4/1, 7.2/1 (w/w)
HeLa MCF-7
(28)
Nitropyrrole-based nucleolipid
siRNA
pUC18 encoding
b-galactosidase
pEGFP-N3
encoding GFP
GAPDH siRNA
1/1, 5/1, 10/1, 15/1, 20/1
(cation/anion mole ratio)
(29)
Glycosyl nucleoside lipid (GNL)
Fluorescein-labelled
oligonucleotide
(ON17-mer)
Plasmid DNA
CHO HepG2
NIH3T3
Huh-7
Hek 293
(38)
diC16dT/DOPE
(10/90 mole ratio)+15 mM Ca2+
pEGFP encoding GFP
4/1 (w/w)
(33)
Cell lines: CHO, Chinese hamster ovarian cells; HepG2, human liver cells; NIH3T3, mouse fibroblast; HeLa, human epitheloid carcinoma cells;
MCF-7, human breast adenocarcinoma cells; Huh-7, human hepatoma cells; Hek 293, human embryo kidney cells. Genes: GFP, green fluorescent
protein; GAPDH, glyceraldehyde-3- phosphate dehydrogenase.
amphiphilic bioconjugate. After cellular uptake, the active
compound must be cleaved from the promoiety to achieve
its therapeutic effect. Covalent coupling to a lipidic molecule may alter the biological activity of the parent drug in
a number of ways (45,46). A lipidic prodrug may improve
the drug oral bioavailability by enhancing its absorption
and/or by decreasing its metabolism. For example,
glycerolipidic prodrugs were conceived with the aim to
enhance lymphatic absorption of polar drugs by mimicking triglycerides metabolism (47). Prodrug may also be
designed to modify the cellular uptake pathway of the
active compound. Amphiphilic conjugates are expected
to enter cells by non-endocytotic mechanism, without
the help of membrane transporters. The pharmacological
activity of the drug may also be prolonged by slow release.
Finally, the prodrug may also decrease toxicity by
achieving the selective delivery or activation of the drug
molecule at the target site.
It must be emphasized that almost all lipophilic prodrugs developed up to now face poor solubility and have
therefore to be encapsulated into a nanocarrier, most often
liposomes, for parenteral administration. Noteworthy, it is
of prime importance that the carrier can retain the
prodrug until reaching its biological target. However,
even a lipophilic anchor does not always ensure the
stable incorporation of the prodrug into phospholipid
bilayers. The size and shape of the molecule and the
relative strengths of interactions between components
can also influence their insertion into the liposome membranes. Low drug loading, poor stability in vivo and
leakage may occur. In contrast, nanosized self-assembled
devices formed by amphiphilic prodrugs have outstanding
advantages including ease of processing, absence of excipients and high drug loading. Furthermore, they carry prospects of improved stability and pharmacokinetics.
Supramolecular organization of prodrug nanoassemblies
is expected to play a key role for the final efficacy of the
drug-delivery system because stability of nanoparticles in
aqueous medium is sensitive to their structure. Moreover,
the ability of these nanoparticles to diffuse into tissues and
to be internalized by cells, their haemolytic activity,
and hence toxicity may be affected by their size, shape
and structure. Two research groups have pioneered the
design of self-assemblies of prodrugs in a systematic way.
Jin and co-workers (48) have synthesized cholesteryl derivatives of antiviral acyclovir, didanosine and zidovudine
nucleoside analogues, involving succinyl, adipoyl or phosphoryl acyl linkers. As shown by TEM, these derivatives
formed nanosized vesicles in water. As far as we know, the
activity of these prodrugs has not yet been evaluated.
The research group of Couvreur (49) has evidenced that
the linkage of squalene, a natural acyclic isoprenoid chain
precursor in the biosynthesis of sterols, to nucleoside analogues led to amphiphilic molecules, which self-organized
in aqueous medium as nanoassemblies of 100–300 nm, irrespective of the nucleoside analogue. Two of these
squalenoyl derivatives, i.e. squalenoyldideoxycytidine
(SQddC) and squalenoylgemcitabine (SQdFdC), have
been studied in detail.
Gemcitabine (dFdC) is a fluorinated cytidine analogue
used in clinic against solid tumours such as pancreas,
non-small cell lung, bladder and breast cancer cell lines.
It is also active against lymphoid and myeloid cancer cell
lines. However, Gemcitabine suffers from serious limitations. Gemcitabine being too hydrophilic to passively
cross the plasma membrane, active transport involving
hENT transporters is required for intracellular drug
uptake. After conversion into its active triphosphate form
by host cell kinases, Gemcitabine is incorporated into
replicating DNA as a false nucleoside, which induces cell
cycle blockage. Down regulation of hENT transporters or
deoxycytidine kinases results in resistance to Gemcitabine
at the cellular level. Moreover, Gemcitabine is rapidly
inactivated in the blood stream by deoxycytidine deaminase, leading to a short plasma half-life (50). A series
of lipophilic prodrugs with increasing length of the hydrophobic chain has been synthesized in order to protect
Gemcitabine from deamination. Valeroyl (C5), heptanoyl
(C7), lauroyl (C12) and stearoyl (C18) fatty acids were
linked to the 4-amino group of Gemcitabine. The metabolic stability in plasma of the C12-dFdC and C18-dFdC
derivatives was significantly enhanced, compared to that
of Gemcitabine. However, these compounds precipitating
in water as large aggregates, needed to be incorporated
Nucleic Acids Research, 2012, Vol. 40, No. 5 1899
into liposomes (or surfactant micelles) for intravenous administration. The encapsulation efficiency of the prodrug
markedly depended on the length of its acyl chain and on
the liposomal composition. A maximum prodrug loading
of 25 mol%, corresponding to 15% w/w drug loading,
was obtained for C18-dFdC. Liposomal formulations of
C12-dFdC and C18-dFdC derivatives exhibited 5- and
2-fold greater cytotoxicity than free Gemcitabine against
HT-29 and KB cell lines, respectively. Moreover, they
improved the drug pharmacokinetics (51).
As explained before, coupling squalene to the sensitive
amine function of Gemcitabine resulted, unlike the
above-mentioned prodrugs, in the spontaneous formation
of nanoparticles 120–140 nm in aqueous medium. As
shown by cryo-TEM and high-resolution synchrotron
SAXS, these nanoassemblies displayed an inverse hexagonal structure, with a lattice parameter of 87.7 Å,
formed by close-packed cylinders whose aqueous cores
were surrounded by hydrophilic Gemcitabine molecules
linked to the squalene moieties. The formation of inverse
hexagonal phase was supported by molecular modelling
(Figure 6) (14). Interestingly, when Gemcitabine was
phosphorylated and further conjugated with squalene,
unilamellar liposomes formed (52).
The SQdFdC nanoassemblies were found impressively
more active than Gemcitabine against many experimental
cancer models, both in vitro and in vivo (53). In vitro,
nanoassemblies displayed higher cytotoxicity than Gem
in murine-resistant leukaemia L1210 10 K cells and in
human leukaemia resistant cell line CEM-ARAC/8C.
In vivo, the SQdFdC nanoassemblies exhibited impressively greater anticancer activity than gemcitabine against
either solid subcutaneously grafted tumours (panc-1,
L1210 and P388) or aggressive metastatic leukaemia (leukaemia L1210, P388 and RNK-16 LGL). The complex
mechanisms resulting in enhanced efficacy of SQdFdC
nanoparticles arose from the better stability of the
prodrug in plasma and a modification of its uptake
pathway. The nanoparticulate form of the prodrug likely
contributed to shield SQdFdC from cleavage and deamination but no direct interaction was found to occur between
nanoparticles and cells. Indeed, it was demonstrated that
nanoparticles did not enter cells by endocytosis, thus
avoiding lysosomal degradation. Mechanistic studies have
evidenced an albumin-enhanced transport of SQdFdC
bioconjugates from nanoparticles to cells across the
aqueous medium. In fact, albumin promoted the dissociation of nanoparticles and mediated the diffusion of
SQdFdC molecules towards the cell membrane where
the prodrug then accumulated. Further partitioning
allowed a dramatic enrichment of SQdFdC into intracellular membranes of the endoplasmic reticulum and endolysosomes (54,55). Consistently with in vitro cell uptake
experiments, combined structural (SAXS–WAXS) and
Figure 6. Self-assembly of SQdFdC. (A) SAXS pattern of the hexagonal phase shown on both linear and log (inset) scales. (B) Schematic
drawing of the inverse hexagonal packing of amphiphilic SQdFdC
molecules. (C) Molecular modelling: face view of two sections of
column each made of six layers of a disk-like assembly composed of
20 conjugates. Molecules are arranged around the aqueous core
Figure 6. Continued
(oxygen atoms/red), with gemcitabine moieties facing inwards
(pyrimidinone nitrogen atoms/blue and fluorine atoms/yellow) and
squalene chains facing outwards. (D) TEM images after freeze
fracture of SQdFdC nanoassemblies [redrawn from Refs (14,15)].
1900 Nucleic Acids Research, 2012, Vol. 40, No. 5
calorimetric studies have highlighted the ability of
SQdFdC molecules to strongly interact with phospholipid
model membranes (56). When fully hydrated DPPC
bilayers were incubated in the presence of SQdFdC
nanoparticles, a slow exchange was indeed evidenced between nanoparticles and DPPC bilayers, confirming the
ability of SQdFdC molecules to be progressively released
from nanoparticles before to concentrate into membranes
(57). In fact, these amphiphilic bioconjugates inserted into
bilayers and affected their curvature, leading to the formation of inverse bicontinuous cubic phases. This interaction may explain why SQdFdC was able to partially
bypass cell resistance to dFdC treatment, when involving
a decrease in the nucleoside transporters expression.
Indeed, contrary to dFdC, SQdFdC was found to exhibit
a significant anticancer activity in CEM/ARAC 8C cells
with deficiency in hENT1 transporters.
When released into the cytoplasm from the membrane
cellular reservoirs, the SQdFdC prodrug amide bond was
found to be cleaved by cathepsin B, an enzyme that can be
hyperexpressed in cancer cells. The progressive release of
dFdC may avoid overloading of intracellular kinases,
which convert Gemcitabine into its active phosphorylated
counterpart. Thus, the prodrug approach could also allow
us to partially overcome another factor of resistance to
dFdC, when intracellular deoxycytidine kinase enzymatic
activity is downregulated, for instance in resistant L1210
10 K cell line. As a whole, SQdFdC nanoparticles enabled
to partially circumvent the three enzymatic or cellular
mechanisms of resistance to Gemcitabine due to hENT1
downregulation, insufficient activity of deoxycytidine kinase or inactivation by deaminases. Furthermore, SQdFdC
nanoparticles appeared to be an attractive delivery system
because of their high deposition in spleen, liver and lungs,
the major metastatic organs in leukemia.
The formation of non-lamellar structures in model
membranes suggested that the amphiphilic properties of
the SQdFdC prodrug could be the source of side effects
in vivo. Rapid hemolysis was indeed evidenced in vitro at
high SQdFdC concentration (57). However, it must be
underlined that preclinical studies did not show such
toxicity in mice. A toxicological profile similar to that of
dFdC was found for SQdFdC (58).
Zalcitabine, formerly called 20 , 30 -dideoxycytidine (ddC),
was approved by the Food and Drug Administration in
1992 for the treatment of the human immunodeficiency
virus (HIV) infection in combination with azidovudine
(AZT). ddC is a synthetic nucleoside analogue of the naturally occurring nucleoside deoxycytidine in which the
30 -hydroxyl group is replaced by hydrogen. Within cells,
ddC is phosphorylated by the sequential action of cellular
kinases. Triphosphorylation allows obtaining dideoxycytidine 50 -triphosphate (ddCTP), the metabolite active
against the virus. ddCTP acts as an inhibitor of HIVreverse transcriptase by competing for utilization of the
natural substrate and a chain terminator by its incorporation into viral DNA. Zalcitabine is well absorbed
showing a bioavailability of 90 % after oral administration. It is weakly metabolized to dideoxyuridine (ddU)
by cytidine deaminase and has a moderate half-life
(t1/2 = 2 h). This low-molecular weight, highly soluble,
nucleoside analogue enters cells mainly via nucleoside
carrier-mediated
(rCNT1
transporter)
pathways.
However, it suffers from limited uptake due to its hydrophilicity and low affinity for nucleoside transporters.
In order to eradicate HIV from infected patients, ddC
should reach latent HIV reservoirs, able to cause reinfection. Effectiveness of ddC is also reduced by emergence of
clinical adverse events including neuropathy, lipodystrophy and pancreatitis. To avoid side effects and
improve ddC efficacy, some attempts have been made to
use liposomes as drug carriers for intravenous administration. Liposome loaded with ddC were prepared but encapsulation efficiency was low (26%) and ddC rapidly
diffused through liposomal bilayers. Several lipophilic derivatives have also been synthesized to improve the therapeutic characteristics of ddC. Different lipophilic moieties
have been attached to the 50 -O- or 4-N-position of ddC.
Three principal classes of ddC prodrugs are known:
ddC-dihydropyridine prodrugs, aiming at increasing the
degree of penetration into the central nervous system,
ddC-4-N-[(dialkylamino)methylene] prodrugs designed to
increase the lipophilicity and stability of the drug, and
ddC masked phosphate prodrugs, designed to deliver the
monophosphate species intracellularly, thus facilitating
the first phosphorylation step. Some of these prodrugs
were found to increase the in vitro activity of ddC or to
decrease its toxicity (46,59,60).
Squalenoylation of ddC (SQddC) yielded spontaneous
formation of nanoassemblies with cubic inner symmetry
upon nanoprecipitation of SQddC in excess water. Two
lattices of space group Pn3m (lattice parameter
a = 77.2 Å) and Ia3d (a = 122.5 Å) were identified using
SAXS. SQddC molecules formed bilayers folded like a
periodic minimal surface, either D (Diamond) or P
(Primitive) surfaces (Figure 7) (15).
When tested in primary cultures of HIV-1-infected
lymphocytes derived from healthy human blood donors,
SQddC exhibited better intracellular penetration and
higher activity. Moreover, since part of the parenterally
administered nanoparticles is taken up by the lymphoid
system, where a significant level of HIV replication occurs,
the SQddC intravenous delivery is expected to improve its
efficacy.
Interestingly, SQdFdC and SQddC, which form hexosomes and cubosomes respectively, differ only by the
geminal fluorines and OH group of Gemcitabine at the
20 - and 30 -positions of the nucleoside. The different supramolecular structures of these squalenoyl amphiphiles can
be explained by the modulation of their spontaneous
monolayer curvature, owing to variation of their interfacial area. The spontaneous monolayer curvature of
SQdFdC, exhibiting a small polar headgroup interfacial
area and a bulky hydrophobic SQ chain, is negative. The
increase in interfacial area, due to the more hydrophilic
headgroup of SQddC, promotes less curved structures like
cubic phases. Similarly, the larger increase in interfacial
area of the amphiphilic compound, induced by the
addition of the 50 -phosphate group to SQdFdC, leads to
the flattening of the monolayers and to the stabilization of
liposomes.
Nucleic Acids Research, 2012, Vol. 40, No. 5 1901
and linked to a lipid moiety. Self-organization of these
new compounds arises from the interplay between aggregation of the lipid chains and base–base interactions.
Subtle differences in molecule chemical structure may
cause variations in the self-assembly pattern.
Nucleolipid supramolecular assemblies are promising
biocompatible nanocarriers for therapeutic molecules.
Specifically, nucleic acids can be confined into lamellar
or hexagonal phases or entrapped in hydrogel networks.
Neutral or anionic nucleolipids are also expected to offer
an alternative to cationic lipids to improve transfection efficacy. The challenge remains to develop safe nucleolipidbased devices displaying high transfection efficiency
in vivo. Noteworthy, self-assembly of oligonucleotides
conjugated with non-cationic lipids could be an efficient
strategy for the delivery of nucleic acids.
Concerning prodrugs, squalenoylation is an innovative
nanomedicine concept, which offers new opportunities to
obtain more potent therapeutic compounds, able to
overcome resistance to the currently available nucleoside
analogues. An attractive feature is that cubosomes or
hexosomes with reproducible mean sizes formed spontaneously, without the need of high-energy dispersing
methods or dispersing agents. The relationship between
the chemical structure of the parent drug, the supramolecular organization of the squalenoyl prodrug and
the therapeutic activity open an exciting way for future
investigations.
FUNDING
The European Research Council under the European
Community’s Seventh Framework Programme FP7/
2007-2013 (249835). Funding for open access charge:
The European Research Council under the European
Community’s Seventh Framework Programme FP7/
2007-2013 (249835).
Conflict of interest statement. None declared.
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Figure 7. Self-assembly of SQddC. (A) SAXS pattern of the Pn3m and
Ia3d inverse bicontinuous cubic phases shown on both linear and log
(inset) scales. The stars denote the reflections of the Ia3d phase. (B)
Schematic drawing of the Pn3m and Ia3d inverse bicontinuous cubic
phases. (C) TEM image after freeze fracture of a SQddC cubosome
[redrawn from Ref. (15)].
In conclusion, it should be emphasized that, besides
Gemcitabine and Zalcitabine, squalenoylation is currently
extended to other nucleoside analogues such as
Didanosine and to polar therapeutic compounds.
CONCLUSION
High-resolution structural investigations reveal the diversity and complexity of assemblies of bioinspired amphiphiles possessing a polar head derived from a nucleobase
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